The protective antigen (PA) of the B.anthracis is the principal protective immunogen of the licensed US and UK human vaccines. It facilitates the transport of Lethal Factor (LF) and Edema Factor (EF) across cell membranes to enable them to exert their toxic effects. By neutralizing the ability of PA to bind to cells and to LF and EF, it is possible to block anthrax toxin activity and prevent morbidity and mortality. As a consequence considerable effort has been directed towards identifying the key T and B cell epitopes of PA to enable a better understanding of the basis of protection and to facilitate the design of smaller, optimised vaccines, which combine efficacy with reduce physiological load, an important attribute when attempting to develop live vector expression system (Flick-Smith et al., 2002). While the central role of antibodies with specificity to PA in protection is well described, the contribution of the LF and EF specific responses in less well defined. It has been reported that antibodies to LF can confer complete protection in mice to lethal toxin challenge (Price et al., 2001) and live agent (Pezard et al., 1995). It has also been reported that the co-administration of LF and PA results in the synergistic enhancement in the polyclonal antibody response to each protein (Pezard et al., 1995; Price et al., 2001). This may be a consequence of the formation of LF/PA complex in the blood resulting in enhanced uptake by antigen presenting cells (Ezzell and Abshire, 1992). The significance of this response in terms of protective efficacy has yet to be determined. We hypothesis that at the protein level interaction of LF with PA enhances the immune response as a consequence of complex formation which results in enhanced uptake by APCs. This synergy raises the possibility of creating a defined vaccine which combines the protective epitopes of PA and LF with the ability to self adjuvantize thus optimizing the resulting antibody response. Current human anthrax vaccines are given by injection and require multiple dosing over 18 months to achieve protection. Bio-terror attacks are likely to occur without warning thus there is an urgent need for user friendly (needle free) formulations capable of invoking protection following minimal dosing in days rather than months. We propose to develop Salmonella based vaccines expressing the key protective epitopes of PA and LF as oral vaccines to facilitate mass vaccination. Product 1. The key protective epitopes of LF. The domains of LF which confer protection against anthrax toxin will be identified, expressed and purified. Product 2. An oral, live anthrax vaccine based on Salmonella enterica serovar Typhi Ty21a, a strain licensed for human use. The level of PA expression will be optimised and a vaccine developed within two years. Product 3. A formulation of PA and LF domains which maximize the immune response against Anthrax Toxin The combination of PA and LF domains which maximize the anthrax toxin specific immune response will be determined. These proteins could be employed as a protein vaccine in addition to being expressed from Salmonella. Product 4. Salmonella enterica serovar Typhi Ty21a expressing the key domains of PA and LF. The combined expression of PA and LF will reduce the number of doses required to stimulate protection (year 4). Product 5. A single dose oral, live anthrax vaccine based on Salmonella enterica serovar Typhi CVD 908-htrA expressing the key domains of PA and LF. (year 4)